Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-09-02
2001-04-03
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S005000, C435S007100, C435S007930, C435S188000, C435S091500, C435S007920, C435S097000, C436S527000, C436S529000, C436S532000, C436S530000, C530S391100, C530S391900, C530S395000, C530S405000, C530S317000, C530S402000, C530S403000, C530S324000, C530S325000, C530S326000, C530S323000, C530S333000, C530S333000, C530S333000, C514S011400
Reexamination Certificate
active
06210908
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to activated peptides, conjugates and methods of preparation thereof, and their use in diagnostic assays and therapeutics.
BACKGROUND OF THE INVENTION
Numerous assays have been developed for the detection and determination of proteins, e.g., antibodies in biological fluids. One class, the immunoassays, which has evolved into an invaluable tool in diagnostics, is based upon the principle of specific binding of antibodies to haptens and/or antigens. In a typical immunoassay, a test sample, containing an analyte of interest, and an antibody, to which it specifically binds, are incubated, and then washed to separate free and bound analytes. An enzyme-labeled antibody that recognizes the resulting complex is added, incubated, and washed, and finally substrate, an enzyme detection system, is added and the labeled complex is detected and determined.
Conjugates of peptides specific to antibodies have been used advantageously in immunoassays. Conjugates of peptide analogues of viral proteins, that is, segments of the proteins bearing the epitopic sequence, for example, of the human immunodeficiency viruses (HIV) and labeled enzymes linked through a maleiimide moiety have been described as being particularly advantageous in the detection and determination of antibodies to HIV. See U.S. Pat. No. 5,294,536 granted to Paul S. Palumbo on Mar. 15, 1994 ('536-patent). The conjugates of activated peptides, prepared by the processes described in the '536- patent are not homogenous, the activated peptides being derived from the terminal amino group and/or the internal amino and hydroxyl groups of the peptide analogue. Interaction of the internal amino and/or hydroxyl groups of the peptide analogue and the crosslinking agent, in addition to that of the terminal amino group, diminishes the effectiveness of the epitopic centers of the peptide analogue thereby reducing the sensitivity of the assay for the detection and determination of specific binding antibodies. For example, interaction of the amino group of the lysine subunit and/or the amino and the hydroxy subunits of the serine subunit of the epotopic segment of the peptide analog of the HIV virus and the crosslinker reduces the sensitivity of the immunoassay for the detection or determination of antibodies to HIV, the greater the interaction of the internal amino and/or hydroxyl group relative to the terminal amino group, the lower the sensitivity of the assay. The processes for the preparation of activated peptides 3 and conjugates 5 described in the aforementioned '536-patent involve condensation of an isocyanatomaleiimide 1
wherein X is a spacer group with a peptide having a terminal amino group of formula 2
RNH
2
2
wherein R is the remainder of the peptide to form an activated peptide 3
wherein R and X are as above, which is then condensed with a thiolated enzyme of formula 4
R
1
SH 4
wherein R
1
is the remainder of an enzyme to form a conjugate of formula 5
Wherein X, R, and R
1
are as defined herein. The critical step in the process, the reaction of the crosslinker 1 with a peptide 2, characterized by the presence of a terminal amino group and internal amino and/or hydroxyl groups, affords an activated peptide 1 derived from the terminal amino group, as well as activated peptides derived from the internal amino and/or hydroxyl groups of the peptide, and combinations thereof.
SUMMARY OF THE INVENTION
It has now been found that by performing the reaction of the isocyanatomaleiimide 1 with a peptide 2 as a salt of a strong protonic acid, the activated peptide is formed substantially free of activated peptides derived from internal amino and/or hydroxyl groups, e.g., of serine or lysine, i.e., the reaction takes place almost exclusively at the terminal amino group of the peptide 2 to regiospecifically form an activated peptide 3 with the epitopic segment of the peptide 2 essentially intact.
It has now also been found that the integrity of the binding region of the activated peptide 3 is maintained in the conjugate 5 and that use of the essentially homogeneous conjugate 5 of the invention in an immunoassay results in a marked improvement of the sensitivity of the assay.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
More particularly, the present invention relates to activated peptides of formula 3
wherein X is loweralkylene, an aromatic carbocyclic moiety or a saturated carbocyclic moiety and R is the remainder of a peptide having a terminal primary amino group and free internal hydroxyl and/or amino groups, useful for the preparation of conjugates 5
wherein R and X are as defined above and R
1
is the remainder of an enzyme having a free thiol group, useful for the detection and determination of antibodies in samples of interest related to the human immunodeficiency viruses.
Preferred activated peptides 3 are those wherein X is an aromatic moiety; most preferred are peptides wherein X is phenyl, and R is the remainder of a peptide analogue having a terminal amino group selected from the group consisting of
As used throughout the specification and appended claims, the term alkylene refers to a saturated straight or branched chain hydrocarbon having 1 to 10 carbon atoms. Examples of alkylene groups are methylene (—CH
2
—), ethylene (—CH
2
CH
2
—), butylene
The term “lower” as applied to alkylene groups refers to a carbon skeleton having 1 to 6 carbon atoms.
The term “alpha-amino acid” refers to a compound characterized by the presence of a carboxylic acid group and an alpha-amino group bound to the same carbon atom
Examples of amino acids are alanine, valine, leucine, isoleucine, proline (or hydroxyproline), phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine.
The term “peptide” refers to polymers of two or more amino acids linked covalently through the carboxyl group of an amino acid and the amino group of another with the elimination of water. Examples of peptides are those compiled herein in the Sequence Listing.
The term “protein” refers to a macromolecule of one or more chains of amino acids bound covalently through peptide bonds
Proteins include enzymes, antibodies, antigens, peptides and the like.
The term “hapten” refers to a low-molecular-weight compound that reacts specifically with an antibody, but does not stimulate antibody production unless complexed with a carrier protein.
The term “antigen” refers to a substance or entity (usually a protein) that induces the direct production of antibodies.
The term “spacer group” refers to a moiety bridging the isocyanato and maleiimides groups and linking the protein and thiolated enzyme moieties of a conjugate. Examples of spacer groups are alkylene (hereindefined), aromatic carbocyclics, such as phenyl or naphthyl optionally having an alkylene group for attachment to the maleiimide moiety and dimethylamino, methoxy, ethoxy, methyl, ethyl, sulfonamido, sulfonic acid substituents, and saturated carbocyclics such as cyclohexyl, cyclohexylalkyl, cyclopentyl, cyclopentylalkyl, cycloheptyl, cycloheptylalkyl.
The term “regiospecific” refers to a process in which one specific structural or positional isomer is formed to the essential exclusion of other possible isomers.
The term “remainder” as applied to peptides refers to the moiety bound to the terminal amino group thereof, for example, the terminal amino group of the terminal glycine moiety of the sequence of amino acids shown below:
The term “remainder” as applied to an enzyme having a free thiol group refers to the moiety bound to the thiol group thereof, for example, specific enzymes such as beta-D-galactosidase, peroxidase, glucose oxidase and alkaline phosphatase, in which a thiol group has been introduced.
The N-terminal activated peptides 3 of the present invention are prepared by contacting an isocyanatomaleiimide 1 with a peptide 2, as a salt of a strong protonic acid, having in the remainder free amino and/or h
Annunziato Michael E.
Palumbo Paul S.
Buchanan Robert L
Buckley Linda M
Cook Lisa V.
Dade Behring Marburg GmbH
Le Long V.
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